IFI44L promoter hypomethylation is associated with a clinically defined immune-inflammatory phenotype in recurrent pregnancy loss

Recurrent pregnancy loss (RPL) is a heterogeneous condition in which immune dysregulation may contribute to pregnancy loss in a subset of patients. Reliable molecular biomarkers for immune-related phenotypes remain limited. IFI44L promoter hypomethylation is an interferon-associated epigenetic feature. This study aimed to evaluate whether IFI44L promoter methylation is associated with clinically defined immune-inflammatory phenotypes in RPL. This retrospective single-center study included a derivation cohort of 119 women with RPL (31 clinically defined immune-inflammatory, 31 non-immune/structural, and 57 unexplained) and a later-enrolled same-center validation cohort of 54 non-overlapping participants. Whole-blood IFI44L promoter methylation was measured by PCR–melting curve analysis. The 33.3% IFI44L promoter methylation cutoff was selected by maximizing the Youden index in the primary 31-versus-31 derivation comparison and was locked before the later-enrolled analysis. Group comparisons, rank correlations, ROC analyses, and exploratory Firth penalized logistic regression were performed. Median IFI44L promoter methylation was 19.0% in the immune-inflammatory group, 51.0% in the non-immune/structural group, and 49.0% in URPL (P < 0.0001). Same-day CBC data were available for all 119 participants; absolute lymphocyte counts were lower in the immune-inflammatory group, but lower methylation remained associated with immune-inflammatory classification in an exploratory medication- and leukocyte-adjusted model. IFI44L methylation was inversely associated with ordered ANA titer (Spearman ρ = −0.553, P < 0.0001) and, more modestly, with the investigator-defined aPL profile category (ρ = −0.265, P = 0.0038). The primary two-group AUC was 0.995 (95% CI, 0.985–1.000), and the secondary immune-versus-all-other RPL AUC was 0.975 (95% CI, 0.954–0.997). The locked 33.3% IFI44L promoter methylation cutoff classified 6/57 (10.5%) URPL participants as IFI44L-low. In the later-enrolled same-center validation cohort, the AUC was 0.897 (95% CI, 0.798–0.997). Whole-blood IFI44L promoter hypomethylation was associated with a clinically defined immune-inflammatory phenotype and autoimmune serological features in RPL. The association remained substantial in exploratory models accounting for recorded medication exposure and routine leukocyte counts. These findings support IFI44L promoter methylation as a promising investigational marker for characterizing immune-related heterogeneity in RPL, while prospective studies are needed to establish its biological basis and clinical utility in larger independent multicenter cohorts.